Salmonella pathogens infect over a billion people each year worldwide resulting in 3 million deaths annually from septicemia, mostly in HIV-infected patients and 700,000 from typhoid fever (W.H.O. estimates). It is one of many gram-negative plant and animal pathogens that have evolved specialized secretion systems, Type III Secretion (TTS) Systems, to facilitate the ordered delivery of virulence effector proteins. The pathogenic TTS systems evolved from the flagellar system, and ensures the efficient, ordered assembly of the bacterial flagellum. The virulence systems have maintained the ordered delivery mechanism of the TTS apparatus to ensure that individual virulence determinants are secreted at the appropriate stage of the infection process. The flagellum serves as a model TTS system for understanding how regulatory mechanisms control the assembly of large structures, and how the TTSS can differentially select substrates for secretion at the appropriate stage of the infection process. We are studying the mechanisms that coordinate the regulation of flagellar gene expression to the assembly of the bacterial flagellum. We have previously shown that one critical regulatory mechanism involves a switch in the secretion substrate specificity of the flagellar TTS apparatus upon hook-basal body (HBB) completion. The secretion of late substrates releases bound secretion chaperones, sigma 28, FlgN, and FliT to initiate gene regulation at the time of HBB completion (checkpoint!). We will determine what targets specific proteins for secretion at their proper assembly time. Specific mechanisms to be investigated include the nature of the flagellar TTS signal, the secretion specificity switch that controls flagellar hook length and the switch in secretion specificity from hook-type substrates to late secretion substrates. We have strong evidence that the FliK protein acts as a molecular ruler to control the final length of the rod-hook complex, and rod-length control is intrinsic to the rod protein FlgG. We will continue to investigate the dual roles for Type III Secretion Chaperones (TTSC) in assembly and gene regulation and in the process of localized translation of secretion substrates at the cytoplasmic base of the flagellum. We will determine the role of the membrane-anchored regulator, Flk, which senses outer membrane penetration and prevents premature secretion of late substrates prior to HBB completion. Because the TTS system is a target for vaccine development against gram-negative pathogens, understanding the process of assembly, secretion and regulation of the flagellar model system will aid in the development of such vaccines.